All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast
Saccharomyces cerevisiae
and neurological or other disorders in humans. In
S
.
cerevisiae
, ...lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G
46
(m
7
G
46
), N
2
,N
2
-dimethylguanosine (m
2,2
G
26
), or 4-acetylcytidine (ac
4
C
12
), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5’-3’ exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast
Schizosaccharomyces pombe
for mutants lacking m
7
G
46
. In contrast,
S
.
cerevisiae trm6/gcd10
mutants with reduced 1-methyladenosine (m
1
A
58
) specifically target pre-tRNA
i
Met(CAU)
to the nuclear surveillance pathway for 3’-5’ exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m
1
A
58
and tRNA
i
Met(CAU)
in both
S
.
pombe
and
S
.
cerevisiae
. We find that
S
.
pombe trm6Δ
mutants lacking m
1
A
58
are temperature sensitive due to decay of tRNA
i
Met(CAU)
by the RTD pathway. Thus,
trm6Δ
mutants had reduced levels of tRNA
i
Met(CAU)
and not of eight other tested tRNAs, overexpression of tRNA
i
Met(CAU)
restored growth, and spontaneous suppressors that restored tRNA
i
Met(CAU)
levels had mutations in
dhp1/RAT1
or
tol1/MET22
. In addition, deletion of
cid14
/
TRF4
in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of
S
.
cerevisiae trm6
mutants revealed a major role of the RTD pathway in maintaining tRNA
i
Met(CAU)
levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m
1
A
58
in the biology of tRNA
i
Met(CAU)
throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The numerous post-transcriptional modifications of tRNA play a crucial role in tRNA function. While most modifications are introduced to tRNA independently, several sets of modifications are found to ...be interconnected such that the presence of one set of modifications drives the formation of another modification. The vast majority of these modification circuits are found in the anticodon loop region where the largest variety and highest density of modifications occur compared to the other parts of the tRNA and where there is relatively limited sequence and structural information. We speculate here that the modification circuits in the anticodon loop region arise to enhance enzyme modification specificity by direct or indirect use of the first modification in the circuit as an additional recognition element for the second modification. We also describe the five well studied modification circuits in the anticodon loop, and outline possible mechanisms by which they may act. The prevalence of these modification circuits in the anticodon loop and the phylogenetic conservation of some of them suggest that a number of other modification circuits will be found in this region in different organisms.
tRNA biology charges to the front Phizicky, Eric M; Hopper, Anita K
Genes & development,
09/2010, Letnik:
24, Številka:
17
Journal Article
Recenzirano
Odprti dostop
tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA ...maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis.
High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications ...interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative methylation analysis using ARM-seq provides the first detailed, transcriptome-scale map of these modifications and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-seq demonstrates that tRNA fragments accurately recapitulate the m(1)A modification state for well-characterized yeast tRNAs and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state and may reveal new roles for tRNA fragments as biomarkers or signaling molecules.
All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, ...lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G.sub.46 (m.sup.7 G.sub.46 ), N.sub.2,N.sub.2 -dimethylguanosine (m.sup.2,2 G.sub.26 ), or 4-acetylcytidine (ac.sup.4 C.sub.12 ), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5'-3' exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m.sup.7 G.sub.46 . In contrast, S. cerevisiae trm6/gcd10 mutants with reduced 1-methyladenosine (m.sup.1 A.sub.58) specifically target pre-tRNA.sub.i .sup.Met(CAU) to the nuclear surveillance pathway for 3'-5' exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m.sup.1 A.sub.58 and tRNA.sub.i .sup.Met(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6DELTA mutants lacking m.sup.1 A.sub.58 are temperature sensitive due to decay of tRNA.sub.i .sup.Met(CAU) by the RTD pathway. Thus, trm6DELTA mutants had reduced levels of tRNA.sub.i .sup.Met(CAU) and not of eight other tested tRNAs, overexpression of tRNA.sub.i .sup.Met(CAU) restored growth, and spontaneous suppressors that restored tRNA.sub.i .sup.Met(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNA.sub.i .sup.Met(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m.sup.1 A.sub.58 in the biology of tRNA.sub.i .sup.Met(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Modification defects in the tRNA anticodon loop often impair yeast growth and cause human disease. In the budding yeast Saccharomyces cerevisiae and the phylogenetically distant fission yeast ...Schizosaccharomyces pombe, trm7Δ mutants grow poorly due to lack of 2'-O-methylation of C32 and G34 in the tRNAPhe anticodon loop, and lesions in the human TRM7 homolog FTSJ1 cause non-syndromic X-linked intellectual disability (NSXLID). However, it is unclear why trm7Δ mutants grow poorly. We show here that despite the fact that S. cerevisiae trm7Δ mutants had no detectable tRNAPhe charging defect in rich media, the cells constitutively activated a robust general amino acid control (GAAC) response, acting through Gcn2, which senses uncharged tRNA. Consistent with reduced available charged tRNAPhe, the trm7Δ growth defect was suppressed by spontaneous mutations in phenylalanyl-tRNA synthetase (PheRS) or in the pol III negative regulator MAF1, and by overexpression of tRNAPhe, PheRS, or EF-1A; all of these also reduced GAAC activation. Genetic analysis also demonstrated that the trm7Δ growth defect was due to the constitutive robust GAAC activation as well as to the reduced available charged tRNAPhe. Robust GAAC activation was not observed with several other anticodon loop modification mutants. Analysis of S. pombe trm7 mutants led to similar observations. S. pombe Trm7 depletion also resulted in no observable tRNAPhe charging defect and a robust GAAC response, and suppressors mapped to PheRS and reduced GAAC activation. We speculate that GAAC activation is widely conserved in trm7 mutants in eukaryotes, including metazoans, and might play a role in FTSJ1-mediated NSXLID.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The life and times of a tRNA Phizicky, Eric M; Hopper, Anita K
RNA,
07/2023, Letnik:
29, Številka:
7
Journal Article
Recenzirano
Odprti dostop
The study of eukaryotic tRNA processing has given rise to an explosion of new information and insights in the last several years. We now have unprecedented knowledge of each step in the tRNA ...processing pathway, revealing unexpected twists in biochemical pathways, multiple new connections with regulatory pathways, and numerous biological effects of defects in processing steps that have profound consequences throughout eukaryotes, leading to growth phenotypes in the yeast
and to neurological and other disorders in humans. This review highlights seminal new results within the pathways that comprise the life of a tRNA, from its birth after transcription until its death by decay. We focus on new findings and revelations in each step of the pathway including the end-processing and splicing steps, many of the numerous modifications throughout the main body and anticodon loop of tRNA that are so crucial for tRNA function, the intricate tRNA trafficking pathways, and the quality control decay pathways, as well as the biogenesis and biology of tRNA-derived fragments. We also describe the many interactions of these pathways with signaling and other pathways in the cell.
Intellectual disability is a common and highly heterogeneous disorder etiologically. In a multiplex consanguineous family, we applied autozygosity mapping and exome sequencing and identified a novel ...homozygous truncating mutation in
PUS3
that fully segregates with the intellectual disability phenotype. Consistent with the known role of Pus3 in isomerizing uracil to pseudouridine at positions 38 and 39 in tRNA, we found a significant reduction in this post-transcriptional modification of tRNA in patient cells. Our finding adds to a growing list of intellectual disability disorders that are caused by perturbation of various tRNA modifications, which highlights the sensitivity of the brain to these highly conserved processes.
Ribosome profiling data report on the distribution of translating ribosomes, at steady‐state, with codon‐level resolution. We present a robust method to extract codon translation rates and protein ...synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate‐reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation.
Synopsis
Ribosome profiling experiments in wild‐type yeast and in mutants with altered tRNA levels illustrate that neither elongation rate nor translational efficiency is affected by tRNA abundance under physiological conditions.
A novel statistical model provides robust inference of codon translation rates and protein synthesis rates and hence better measures translation efficiency.
Codon translation rates have insignificant correlation with measures of codon bias.
Direct experimental manipulation of tRNA abundance does not affect elongation rates on affected codons or translation efficiency of overall genes.
Other sequence signals, such as mRNA structure and an initiation sequence motif, correlate to translation efficiency and may be causal determinants.
Ribosome profiling experiments in wild‐type yeast and in mutants with altered tRNA levels illustrate that neither elongation rate nor translational efficiency is affected by tRNA abundance under physiological conditions.
Despite the universality of tRNA modifications, some tRNAs lacking specific modifications are subject to degradation pathways, while other tRNAs lacking the same modifications are resistant. Here, we ...suggest a model in which some modifications have minor, possibly redundant, roles in specific tRNAs. This model is consistent with the low specificity of some modification enzymes. Limitations of this model include the limited assays and growth conditions on which these conclusions are based, as well as the high specificity exhibited by many modification enzymes with important roles in translation. The specificity of these enzymes is often enhanced by complex substrate recognition patterns and sub-cellular compartmentalization.
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